Association between serum 25‐hydroxyvitamin D level and inflammatory markers in hemodialysis‐treated patients

Abstract Objective To investigate the relationship between serum 25‐hydroxyvitamin D (25(OH)D) level with novel inflammatory markers in hemodialysis‐treated patients. Methods A total of 167 maintenance hemodialysis‐treated patients were enrolled in this cross‐sectional study. The patients were divided into vitamin D deficiency (a serum 25(OH)D level <20 ng/mL) and nondeficiency (a serum 25(OH)D level ≥20 ng/mL) groups. The neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and monocyte to lymphocyte ratio (MLR) were calculated by the complete blood cell count. The relationship between 25(OH)D level with other parameters was assessed by bivariate correlation analysis and linear regression analysis. Results There were significant differences between the two groups in terms of age, diabetes, levels of albumin, creatinine, high‐density lipoprotein cholesterol (HDL‐C) and low‐density lipoprotein cholesterol (LDL‐C) as well as NLR and MLR (p = .004, p = .031, p < .001, p = .043, p = .008, p = .006, p = .002, and p < .001, respectively). There exist negative correlations between serum 25(OH)D level with age, diabetes, alkaline phosphatase level, NLR, PLR, and MLR (p = .002, p = .002, p = .037, p = .001, p = .041, and p < .001, respectively) and positive correlations between serum 25(OH)D level with albumin level, creatinine level, phosphorus level, HDL‐C, and LDL‐C (p < .001, p < .001, p = .013, p = .02, p = .002, respectively). Multiple analysis results showed that sex, diabetes, albumin level and NLR were independently associated with serum 25(OH)D level (p = .021, p = .015, p = .033, and p = .041, respectively). High values of NLR and MLR were associated with patients with serum 25(OH)D deficiency. There were negative interplays between serum 25(OH) D level with NLR, PLR, and MLR and also an independent association between serum 25(OH) D level with NLR. Conclusion Collectively, serum 25(OH)D level has a negative correlation with inflammatory markers.

Patients with chronic kidney disease (CKD) including end-stage renal disease (ESRD) are prone to vitamin D deficiency.The prevalence of vitamin D deficiency or insufficiency can reach over 80%. 1,2Of note, vitamin D deficiency is implicated in cardiovascular events and all-cause mortality in hemodialysis-treated patients. 3,4he mortality risk in patients with CKD is decreased by 14% for every 10 ng/mL increase in 25-hydroxyvitamin D (25(OH)D) level, 5 while being increased by 30% for 25(OH)D level lower than 18 ng/mL. 6icroinflammation occurring in ESRD patients causes atherosclerosis, malnutrition and cardiovascular disease, and is responsible for increased mortality of these patients. 7In the context of the confirmed relationship between vitamin D and inflammation, a clear consensus has not been reached.The neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and monocyte to lymphocyte ratio (MLR) are accessible, inexpensive, and reproducible markers for detecting inflammation recently.Neutrophils are the main effector cells during the systemic inflammatory response, which, together with other inflammatory cells, can be used to mediate the early stage of infection as having proinflammatory properties, and NLR is usually characterized by an increase in neutrophils and a decrease in lymphocytes. 8Platelets are rich in proinflammatory agents, and the interaction of platelets with T lymphocytes mediated by P-selectin reduces lymphocyte proliferation, leading to diminished proinflammatory cytokines such as interferon-α (IFN-α), tumor necrosis factor-α (TNF-α), and interleukin (IL)-17, and elevated anti-inflammatory cytokines such as IL-10. 9Besides, increased MLR is strongly associated with the risk of new-onset CKD in people. 10However, there are limited data on the association between vitamin D and these novel inflammatory markers such as NLR and PLR, and no studies evaluating the interplay between vitamin D and MLR in hemodialysistreated patients.Therefore, this study concentrated on investigating the correlation between serum 25(OH)D level and novel inflammatory markers in hemodialysistreated patients.

| Study design
This was a cross-sectional study performed on 187 ESRD patients treated with conventional maintenance hemodialysis from January 2022 to December 2022 in our hospital.The inclusion criteria were patients aged over 18 and receiving treatment of maintenance hemodialysis (4-h dialysis regimens for twice to thrice per week) for at least 3 months.The exclusion criteria were set as follows: patients having no data on serum 25(OH)D level but a history of malignancy, receiving peritoneal dialysis, accompanied with acute infection (within 2 weeks before enrollment, whether controlled or not) and active inflammatory disease, and receiving treatment of immunosuppressive drug.Overall, 20 patients were excluded, involving five without data of serum 25(OH)D level, two receiving treatment of peritoneal dialysis, six with acute infection, four with malignancy, two with active inflammatory disease, and one being administered with immunosuppressive drug.Ultimately, the data of 167 patients who met the criteria were included in the study, which was approved by the ethics committee of the First Affiliated Hospital of Ningbo University (2023-042RS) and abided by the Declaration of Helsinki.

| Data collection
Clinical and demographic characteristics of the patients (age, sex, presence of diabetes mellitus, body mass index (BMI), dialysis duration, type of vascular access, dialysis frequency and Kt/v index) were recorded at the time of enrollment.
Fasting venous blood samples were collected directly from patients before connection of the dialysis tube by hemodialysis specialist nurse on dialysis day, and then transported to the central laboratory of the hospital immediately for laboratory analysis.
Laboratory data including serum 25(OH)D level, complete blood count with automated differential counts (involving white blood cell [WBC], neutrophils, lymphocytes, and monocytes), levels of high-sensitivity C-reactive protein (hsCRP), hemoglobin, albumin, creatinine, calcium, phosphorus, intact parathyroid hormone, and lipid parameters were obtained from the medical record database.NLR, PLR, and MLR were calculated.
According to the Kidney Disease Outcome and Quality Initiative guidelines, vitamin D deficiency is defined as a serum 25(OH)D level <20 ng/mL, insufficiency as a serum 25(OH)D level from 20 to 30 ng/mL, and sufficiency as a serum 25(OH)D level >30 ng/mL. 11herefore, patients in this study were separated into vitamin D deficiency group (a serum 25(OH) D level of <20 ng/mL) and nondeficiency group (a serum level of ≥20 ng/mL).

| Statistical analysis
Statistical analyses were carried out using the SPSS 26.0 (IBM Corp.).Descriptive statistics were determined for each variable.Mean ± standard deviation was used to describe continuous variables having normal distribution and median (P25, P75) with asymmetric distribution.The independent samples t-test and the Mann-Whitney U test were applied for analyzing continuous variables.Categorical variables analyzed by χ 2 test were expressed as percentages.Associations between the variables were explored using Pearson's rho (for normally distributed variables) and Spearman's rho (for asymmetrically distributed variables).Linear regression analysis was employed to define independent variables associated with serum 25(OH)D level.A p < .05 was considered statistically significant.

| RESULTS
A total of 167 maintenance hemodialysis-treated patients were included in this study, with the median age of 66 (55-76) years, and 65.3% (n = 109) of males.The mean 25(OH)D level was 25.16 ± 10.08 ng/mL.There were 56 (33.5%) patients in vitamin D deficiency group and 111 (66.5%) patients in nondeficiency group.Only 49 patients (29.3%) had a sufficient vitamin D level.The mean serum 25(OH)D level was 14.88 ± 3.56 ng/mL in the deficiency group and 30.34 ± 8.14 ng/mL in the nondeficiency group.The demographics as well as the clinical and laboratory characteristics of the two groups are displayed in Table 1.There were statistically prominent differences between two groups in terms of age, diabetes, levels of albumin, creatinine, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) as well as NLR and MLR (p = .004,p = .031,p < .001,p = .043,p = .008,p = .006,p = .002,and p < .001,respectively).Although PLR was higher in patients with 25(OH)D deficiency, the difference was not statistically significant (p = .075).hsCRP and other parameters were barely different in the two groups.

| DISCUSSION
Vitamin D is a fat-soluble steroid and plays a central role in bone and mineral metabolism through its hormonal form 1,25-dihydroxyvitamin D, which is produced by 1-alfa hydroxylase in the kidney.Recent data revealed a notably wide tissue distribution of vitamin D receptor and 1-alfa hydroxylase in the same or neighboring cells. 12Decreased circulating 1,25-dihydroxyvitamin D is common in ESRD patients due to the loss of renal 1-alfa hydroxylase.Thus, the external pathway enabling the transformation of 25(OH)D into 1,25-dihydroxyvitamin D via 1-alfa hydroxylase is a contributing factor for many nonclassical actions of vitamin D which takes effect in ESRD patients.It is estimated that over 85% of serum 25(OH)D is utilized by local target tissues for autocrine and paracrine activation of 1,25-dihydroxyvitamin D, 13 thus impacting on inflammatory process in different tissues. 14Besides, a previous study showed that vitamin D deficiency is a highly prevalent and modifiable risk factor in patients with CKD, contributing to inflammation. 15Therefore, vitamin D supplementation is pivotal for patients with ESRD.
A study has confirmed the significant association between serum 25(OH)D level with age, diabetes mellitus, albumin level, and creatinine level.The linear regression analysis results proved that sex, diabetes mellitus, and albumin level are independently related to 25(OH)D level, while age, sex, race, diabetes, current smoking, and BMI are all independently associated with 25(OH)D deficiency in the general population. 16Age 17 and diabetes 18 are the determinants for predicting 25(OH)D level in CKD patients.In hemodialysis-treated patients, it was documented that 25(OH)D level is linked to age, sex, and diabetes but not to albumin level. 19,20A previous report showed that patients with a decreased 25(OH)D level have a lower albumin level, but the difference was not significant. 21Bansal et al. 22 found a weak correlation between 25(OH)D level with weight, sex, levels of hemoglobin, albumin, and alkaline phosphatase, and diabetes in hemodialysis-treated patients.All these studies of dialysis-treated patients did not mention the relationship between vitamin D level and creatinine level.Yildirim et al. 23 reported that creatinine level is markedly lower in 25(OH)D-deficient patients   23 studied the associations between 25(OH) D level with CRP, WBC, and erythrocyte sedimentation rate in the general population and nondialysis-treated CKD patients, and found no relationship between 25(OH)D level and CRP.Several other studies of hemodialysis-treated patients indicated that 25(OH)D level is inversely correlated with CRP. 19,21,24In contrast to these studies, although the median of CRP values was higher in patients with 25(OH)D deficiency in our study, the difference was not statistically significant and there was no prominent correlation between CRP and 25(OH) D level, which may be due to individual characteristics of the studied population.The values of CRP in our cohort were much lower compared to the studies mentioned above.Intriguingly, NLR, PLR, and MLR were all positively correlated with CRP in our study.Furthermore, in terms of inflammation determination, either NLR 25,26 or PLR 27,28 has been confirmed to be the superior one by different studies.CRP in combination with NLR can reduce the CRP cutoff point for distinguishing between infectious and noninfectious inflammation in hemodialysis-treated patients. 29Our study unveiled that NLR had a stronger correlation with CRP than PLR and MLR.NLR might be better for identifying inflammation in hemodialysis than PLR and MLR.
NLR, PLR, and MLR are not only used to determine inflammation, [30][31][32] but also considered to be predictors of all-cause and cardiovascular mortality [33][34][35][36][37][38][39] in ESRD patients and dialysis-treated patients.Numerous studies have compared the roles of NLR, PLR, and MLR in predicting cardiovascular and all-cause mortality in hemodialysis-treated patients.Catabay et al. 40 found that NLR, instead of PLR, is a mortality predictor.Yaprak et al. 28 reported that both NLR and PLR are associated with all-cause mortality in hemodialysis-treated patients but only PLR can independently predict all-cause mortality.Zhang et al. 41 identified that the high NLR value is associated with high all-cause mortality while PLR is a predictor of cardiovascular mortality.Xiang et al. 42 unraveled that high value of MLR strongly and independently predicts all-cause and cardiovascular mortality compared to NLR.
The relationship between NLR and PLR with vitamin D in hemodialysis-treated patients has been less studied.In line with previous data, NLR is obviously higher in the hemodialysis-treated patients with 25(OH)D < 10 ng/mL and is inversely correlated with 25(OH)D level. 43Kara et al. 19   linear regression analysis results verified that NLR was independently associated with serum 25(OH)D level after adjustments for other factors.Thus, the function of NLR was superior to those of MLR and PLR in our study.However, the mechanism of decreasing inflammatory when serum 25(OH)D levels still unclear.Previous study showed that 25(OH)D affects proinflammatory markers IL-1β, IL-6, IL-8, TNF-α via nuclear factor kappa-B and mitogen-activated protein kinase signaling pathways. 44oreover, 25(OH)D inhibited B-cell activation and immunoglobulin synthesis, reduced Th1 and Th17 cells and the production of IFN-γ, IL-17, IL-6, IL-23, and IL-2, and increases Th2 cells, thereby mediating cytokines and TNF-α to limit the inflammatory process. 45Base on this, it is reasonable to suspect that the possible role of 25(OH) D in ESRD can be explained by its role in the regulation of immune cells and inflammatory pathways, which need more experiments to verify.
To the best of our knowledge, this is the first study to determine the relationship between 25(OH)D with MLR, NLR, and PLR in hemodialysis-treated patients.Nonetheless, our study has several limitations.First, this was a cross-sectional study which cannot establish a causal relationship between serum 25(OH)D level and inflammatory markers.Second, biochemical measurements were only performed once.Time-and season-dependent changes of 25(OH)D level were not investigated.Thirdly, the impact of therapeutic interventions was not considered.Lastly, the sample size was relatively small, although the number of patients was adequate to show the statistical significance.

| CONCLUSIONS
Our study demonstrates an inverse association of serum 25(OH) D level and inflammatory markers.Further, large-scale, prospective, and interventional studies are needed to confirm our results and to deeply investigate the associations between serum 25(OH) D level with inflammatory markers in hemodialysis-treated patients.

DATA AVAILABILITY STATEMENT
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

T A B L E 1
Demographics and clinical and laboratory parameters of hemodialysis-treated patients according to serum 25(OH)D levels.Parameters 25(OH)D < 20 ng/mL (n = 56) 25(OH)D ≥ 20 ng/mL (n = 111) p-Value Age (years) 72.0 (60.0, 78.8) 65.0 (53. found that NLR is evidently higher in the 25(OH) D deficiency group in comparison with the normal group.Serum 25(OH)D level is inversely correlated with NLR and PLR, but when determined by linear regression analysis, sex is the only variable significantly associated with 25(OH)D level.In Villafuerte-Ledesma et al.'s 21 study, NLR and PLR are both higher in the presence of 25(OH)D deficiency, manifesting the inverse correlation between NLR and PLR with 25(OH)D level.There was no study investigating the interplay between 25(OH)D and MLR in hemodialysis-treated patients.Our study corroborated that NLR and MLR were both noticeably higher in hemodialysis-treated patients with 25(OH)D deficiency and had a statistically significant negative correlation with 25(OH)D level.Moreover, multiple

T A B L E 3
Correlations between serum 25(OH)D level with other relevant parameters.
T A B L E 2 Bivariate correlations between serum 25(OH)D level with other parameters.